Expression of foreign gene sequences using mammalian hosts is, by now, well-known in the art. Mammalian expression systems are often favored because the host cell possesses processing capability which permits modification of gene products, for example by glycosylation or hydroxylation, unlike bacterial, or even yeast systems.
Also, the ability of mammalian systems to secrete certain gene products efficiently into the medium results in easier harvest and purification. Because the secreted product must be purified from other proteins in the medium, it is clearly desirable to employ hosts capable of growth in defined media--i.e., media free from added proteins. While most mammalian cell lines require supplements, such as serum proteins, Chinese Hamster Ovary (CHO) cells can be maintained in defined, serum-free and protein-free medium. (See Hamilton, W. G. and Ham, R. G. In Vitro (1977) 13: 537-547). CHO cells are also fast growing and well characterized, free of recognizable dangers, and are thus, in addition to their ability to be maintained serum free, ideal hosts for recombinant protein production.
Mammalian expression, of course, requires compatible control sequences. The most commonly employed control sequences, in particular, promoters, have been viral promoters, most prominently the SV40 promoter (see, for example, EPO Publication 108,667, published 16 May 1984; McCormick, et al, Molec Cell Biol (1984) 4: 166-172) or the gene's own promoter, if compatible (U.S. Pat. No. 4,399,216 to Axel, et al). In general, such promoters are less than satisfactory because they cannot be regulated by environmental factors. Absent such control, the linked coding sequences may be expressed at too high or too low a level in the host organism, or at an improper time, and the worker in the art is powerless to control these aspects of expression. Therefore, attempts have been made to utilize mammalian-compatible promoters susceptible to environmental control. Notable among these promoters are the metallothionein promoters, which natively control the expression of metallothionein proteins--proteins which bind tightly to heavy metals. In their native state, the metallothionein promoters are induced both by the presence of heavy metals, for example, cadmium or mercury, and also by steroid hormones and related materials, such as dexamethasone or other glucocorticoids. However, it has not been possible to carry out induction in the absence of protein-supplemented media, thus limiting the use of such promoters to conditions where purification of the protein produced is complicated by the presence of large amounts of additional contaminants.
Various members of the metallothionein promoter family have been used to control expression in mammalian systems. For example, PCT application WO 84/02534, published July 5, 1984, to Hamer, et al, discloses the use of the mouse metallothionein-I (mMT-I) promoter to control the expression of human growth hormone in mouse kidney C127 cells (see also Pavlakis, G. N., et al, Proc Natl Acad Sci (U.S.A.) (1983) 80: 397-401). Brinster, et al, Nature (1982) 296: 39-42, obtained thymidine kinase production in injected mouse embryos under control of the murine MT-I promoter. Karin, M., et al, DNA (1984) 3: 319-325; Nature (1984) 308: 513-519 have studied transient expression under the control of the human metallothionein-II.sub.A system (hMT-II.sub.A) in NIH-3T3 cells, by fusing the hMT-II.sub.A promoter to the coding sequences of thymidine kinase to generate hMT-TK chimeric genes, and monitoring expression of TK after deletions in the promoter sequences. Induction was obtained using either cadmium ion, or, in some instances, dexamethasone; and the locations of these regulatory sites were determined by deletion studies.
None of the expression systems disclosed, including those utilizing some form of the MT promoter, permit satisfactory culturing and induction of the host to produce the desired gene product in continuous high yield and in easily recoverable form, free of added serum or other proteins. The expression system of the present invention permits continuous induction of high levels of expression in hosts grown in serum-free medium, and thus permits high levels of protein production and easy purification of secreted products.
In addition to providing non-toxic induction and culture conditions favorable to purification of the desired protein from the medium, it is desirable to enhance the level of production of this protein. Three general approaches relevant to the invention herein have been used to enhance protein production in the past: inclusion of viral enhancers in the expression system, selection of transformed cells for high levels of expression of the transforming DNA, and amplification of the expression system to increase copy number.
Enhancers are cis-acting DNA elements which stimulate transcription. Their activity is relatively independent of their 5'-3' orientation, and, while position dependent to some degree, is retained over distances as long as several thousand nucleotides. Enhancers have been identified in a number of viral genomes and in specialized cellular genes, such as those responsible for the production of immunoglobulins. The enhancer used in the illustration below, which is derived from the simian virus SV40, has been characterized in some detail (Gruss, P., et al, Proc Natl Acad Sci (USA) (1981) 78: 943-947; Benoist, C., et al, Nature (1981) 290: 304-310; Mathis, D. J., et al, ibid, 310-315). Wasylyk, B., et al, Nucleic Acids Res (1984) 12: 5589-5608 showed that the 72 bp repeat which is thought to be the essential element of the SV40 enhancer has a biphasic dependence on distance from tandem conalbumin promoters linked to the coding sequence for early T antigen used as a diagnostic for transcription enhancement. The invention herein, in some of its aspects, utilizes the stimulating effect of enhancer sequences to increase production levels.
Use of the ability of DHFR to select and to amplify in response to certain drugs to effect an increase in the production of protein by a co-transforming expression system has been practiced for several years. See, for example Kaufman, R. J., et al, Mol Cell Biol (1985) 5: 1750-1759. It is believed that relevant selection for high production of desired protein through selection for drug resistance and co-amplification of the desired gene along with DHFR occurs because the DHFR gene and the co-transforming gene are integrated in nearby positions into the chromosome of the host. Thus, local conditions favoring expression are applicable, in general, to both genes. Further, amplification in response to the drug occurs over distances of approximately 200 kb, thus carrying the co-transforming gene along with the DHFR in making multiple copies.
The MT gene has not been used analogously. While the ability of the MT gene to amplify in response to cadmium ion is known, this has been studied using bovine papilloma virus (BPV), a self-replicating transforming system, and therefore use of this amplification to co-amplify a desired expression system has not been suggested (Karin, M., et al, Proc Natl Acad Sci (USA) (1983) 4040-4044); nor has cadmium resistance been used as a selectable marker when integrated into the gene to identify high level expressors. In some aspects, the invention herein employs an expression system which takes advantage of an MT gene co-transformed with the expression system either to select for transformants with high level expression abilities for the desired sequence, or to amplify the MT and expression sequences simultaneously, or both.
The various aspects of the invention for improving the quality and quantity of expression in CHO cell hosts are illustrated below for the production of a variety of proteins. In one such illustration the gene for human growth hormone is employed, as this material is of practical interest, of benefit therapeutically, and has not been produced in natural form according to current methods. Recombinant production of hGH, although commonly done, does not result in a product which is true to the mixture of materials produced by the pituitary. Native hGH is a mixture which contains approximately 10% of a minor form, 20 kD hGH, in addition to 90% of the major, 22 kD, species. Both species are encoded by a single gene and result from translation of two different mRNAs produced by differential splicing of the second intron from the primary transcript (DeNoto, et al, Nucleic Acids Res (1981) 9: 3719). Hence, production employing cDNA for hGH (EPO Application 108,667 (supra)) or production employing a combination of cDNA and genomic sequences, but lacking the second intron (Hamer, WO84/02534; Pavlakis, Proc Natl Acad Sci (supra)) is not satisfactory from this viewpoint, since only the 22 kD form is produced. Even less satisfactory are bacterially produced recombinant hGH preparations (Goeddel, D. V., et al, Nature (1979) 281: 544-548; Martial, J. A., et al, Science (1979) 205: 602-607) as the hGH thus produced is not secreted into the medium, and at least a major proportion of the hGH produced contains an N-terminal methionine as a result of the recombinant construction and the inability of the host cell further to process the resulting protein.
Significant advantages over, for example, bacterial expression are also seen for hASP. Isolation of hASP from an alveolar proteinosis patient gives a mixture wherein the major species is a 32 kd protein; this indicates that the putatively native form is glycosylated (White, R. T., et al, Nature (1985) 317: 361-363). This is also true of canine ASP where similar heterogeneity (28 kd-36 kd) is found. Human cDNA shows an open reading frame encoding 248 amino acids; the sequence beginning at amino acid 21 encoded by this cDNA corresponds to the 22 N-terminal amino acids of the 32 kd protein isolated from lavage fluid. The cDNA has also permitted location of the exon regions of the gene. The cDNA sequence encodes a number of collagen like Gly-X-Y repeats which contain a proline residue. The presence of hydroxyproline in the native sequence indicates these are hydroxylated. Thus, reconstruction of "native" ASP requires at least two post-translational steps available only in mammalian systems--hydroxylation of the proline residues and glycosylation.
Apolipoproteins AI and AII, found in connection with lipids as carriers in the bloodstream are encoded as preproproteins and secreted in "pro" form. However, the protein associated with phospholipids to generate the stacked disc structure of the lipoprotein fractions is mostly mature protein (Boganouski, D., et al, J Lipid Res (1985) 26: 185). In this case, too, post-translational modifications of which only mammalian cells are capable, are desirable to generate the native functional form.
The present invention provides not only human growth hormone preparations similar to those produced by the pituitary, human ASP preparations similar to those found in lung lavage fluid, and apolipoproteins functionally associated with phospholipids, but also a means for efficient production and recovery of any desired foreign protein under favorable, high production, easy purification conditions.